Ten broccoli [Brassica oleracea L. (Botrytis Group)] accessions were grown in several environments to estimate glucosinolate (GS) variability associated with genotype, environment, and genotype × environment interaction and to identify differences in the stability of GSs in broccoli florets. Significant differences in genetic variability were identified for aliphatic GSs but not for indolyl GSs. The percentage of GS variability attributable to genotype for individual aliphatic compounds ranged from 54.2% for glucoraphanin to 71.0% for progoitrin. For total indolyl GSs, the percentage of variability attributable to genotype was only 12%. Both qualitative and quantitative differences in GSs were detected among the genotypes. Ten-fold differences in progoitrin, glucoraphanin, and total aliphatic GS levels were observed between the highest and lowest genotypes. Only two lines, Eu8-1 and VI-158, produced aliphatic GSs other than glucoraphanin in appreciable amounts. Differences in stability of these compounds among the cultivars were also observed between fall and spring plantings. Results suggest that genetic factors necessary for altering the qualitative and quantitative aliphatic GS profiles are present within existing broccoli germplasm, which makes breeding for enhanced cancer chemoprotectant activity feasible.